Hybridization of plants is recognized as an important process for producing offspring having a combination of the desirable traits of the parent plants. The resulting hybrid offspring often have the ability to outperform the parents in different traits, such as in yield, adaptability to environmental changes, and disease resistance. This ability is called "heterosis" or "hybrid vigor". As a result, hybridization has been used extensively for improving major crops, such as corn, sugarbeet and sunflower. For a number of reasons, primarily related to the fact that most plants are capable of undergoing both self-pollination and cross-pollination, the controlled cross-pollination of plants without significant self-pollination, to produce a harvest of hybrid seeds, has been difficult to achieve on a commercial scale.
In nature, the vast majority of crop plants produce male and female reproductive organs on the same plant, usually in close proximity to one another in the same flower. This favors self-pollination. Some plants, morphology of their reproductive organs which favors cross-pollination. These plants produce hybrid offspring with improved vigor and adaptability. One such morphology in Cannabis ssp. (hemp) involves male and female reproductive organs on separate plants. Another such morphology in Zea mays (corn) involves male and female reproductive organs on different parts of the same plant. Another such morphology in Elaeis quineensis (oilpalm) involves male and fertile female gametes which become fertile at different times in the plant's development.
Some other plant species, such as Ananas comosus (pineapple), favor cross-pollination through the particular physiology of their reproductive organs. Such plants have developed a so-called "self-incompatibility system" whereby the pollen of one plant is not able to fertilize the female gamete of the same plant or of another plant with the same genotype.
Some other plant species favor cross-pollination by naturally displaying the so-called genomic characteristic of "male-sterility". By this characteristic, the plants' anthers degenerate before pollen, produced by the anthers, reaches maturity. See: "Male-Sterility in Higher Plants", M. L. H. Kaul, 1987, in: Monographs on Theoretical and Applied Genetics 10, Edit. Springer Verlag. Such a natural male-sterility characteristic is believed to result from a wide range of natural mutations, most often involving deficiencies, and this characteristic cannot easily be maintained in plant species that predominantly self-pollinate, since under natural conditions, no seeds will be produced.
Some types of naturally occurring male-sterility are cytoplasmatically encoded, while other are nuclear encoded. One type of male-sterility is the result of a combination of both nuclear encoded male-sterility and cytoplasmatically encoded male-sterility. The male-sterility inducing nuclear alleles are usually recessive, and only plants that contain the male-sterility cytoplasmic allele and that are homozygous for the male-sterility inducing nuclear allele are phenotypically male-sterile. In this type of plant, corresponding dominant male-fertility inducing alleles or "fertility restorers" produce a male-fertile phenotype. As a result, the male-sterile offspring of this type of plant can be made male-fertile by pollinating the male-sterile plants with pollen containing the fertility restorers. As a result, the offspring of plants of this type are of commercial value where the economic product is seeds (e.g., for plants such as corn, sorghum and sunflower).
Most of the known naturally occurring male-sterility genes and their corresponding fertility-restorer genes have not been used in breeding or production of new varieties for essentially two reasons: a) insufficient quality of the genes responsible for the male-sterility and restoration characteristics; and b) low cross-pollination capability of the crops in which they occur.
1. The Quality of the Genes
To realize the full potential of a male-sterility/fertility-restorer system, several quality requirements have to be achieved:
a) Stability of the genes encoding the male-sterility under a broad range of different environmental conditions. Most of the currently known systems, whether they are nuclear or cytoplasmatically encoded, do not display sufficient stability. As a consequence of this, under some unpredictable climatological conditions, self-pollination occurs within the plants, and heterogeneous offspring are harvested. According to seed certification requirements, not more than 1% of non-hybrid seed is tolerated for most major field crops. PA1 b) No side effects on the plants. Many cytoplasmic male-sterility genes induce a decrease in plant vigor. This can be tolerated up to a certain level, if the hybrid vigor effect offers a significant improvement of the crop compared to the negative effect. Another side effect which has been observed in crops carrying male-sterility genes consists of an enhanced sensitivity to some plant pathogens (e.g., corn plants carrying T-cytoplasmic male-sterility are highly susceptible to Helminthosporium maydis infections). PA1 (a) a fertility-restorer DNA encoding a first RNA, protein or polypeptide which, when produced or overproduced in a cell of a flower, particularly a male or female organ thereof, a seed or an embryo of the plant, prevents the activity in the flower, seed or embryo cell of a second RNA, protein or polypeptide that, when produced or overproduced in the flower, seed or embryo cell, could otherwise significantly disturb the metabolism, functioning and/or development of the flower, seed or embryo cell; the second RNA, protein or polypeptide being encoded by a sterility DNA that is also stably integrated into the nuclear genome of the plant and is under the control of a sterility promoter capable of directing expression of the sterility DNA selectively in specific cells of each of the plant's flowers, particularly a male or female organ thereof, and/or seeds and/or embryos and of thereby rendering the plant male-sterile or female-sterile in the absence of expression of the fertility-restorer DNA in the specific flower, seed and/or embryo cells; and PA1 (b) a first promoter capable of directing expression of the fertility-restorer DNA at least in the specific flower, seed and/or embryo cells of the plant where the sterility promoter directs gene expression of the sterility DNA; the fertility-restorer DNA being in the same transcriptional unit as, and under the control of, the first promoter. PA1 (c) a first marker DNA encoding a third RNA, protein or polypeptide which, when present at least in a specific tissue or specific cells of the plant, renders the plant easily separable or distinguishable from other plants which do not contain the third RNA, protein or polypeptide at least in the specific tissue or specific cells; and PA1 (d) a second promoter capable of directing expression of the first marker DNA at least in the specific tissue or specific cells; the first marker DNA being in the same transcriptional unit as, and under the control of, the second promoter.
Restorer genes also often display negative side effects although these are usually not due to the genes themselves but to genes closely linked to the restorer genes. These side effects consist, in most cases, of an increased disease or pest susceptibility or a decreased quality of the crop.
2. Efficiency of cross-pollination
Reasonably efficient Cross-pollination is essential for the production of hybrid seeds at an acceptable cost. For major field crops that are poorly adapted to cross-pollination, it is unrealistic to assure crops-pollination by hand. Therefore, it has been envisaged to sell, as a commercial product, not the F1 hybrid, but the selfed F.sub.2 offspring thereof (e.g., cotton and wheat). The disadvantage of this method lies, however, in the loss of homogeneity and heterosis and the segregation of specific useful gene combinations. To assure high yield of a crop by a farmer, it is advantageous that hybrid crops be fully fertile (with the exception of very efficient cross-pollinating species such as corn and oilseed rape). This is particularly the case with crops that form heavy or sticky pollen which is not easily transported by wind (e.g., cotton), with crops that are not attractive to pollinating insects (e.g., wheat) and with crops which display cleistogamy (e.g., soybean).